Much of the analytical drive in biological research areas results from the desire to understand biological systems and to develop therapeutic agents for treating disease. Increasingly, worldwide research is focused on obtaining an understanding of protein pathways in organisms, and on the correlation between protein modification and disease. Current thinking in biology avers that manifestation of disease states ultimately involves the interaction of proteins in a manner that differs from non-disease tissue. The interest in understanding protein function is therefore becoming prevalent in all biologically relevant fields, including proteomics, biotechnology, drug discovery, and molecular diagnostics.
The study of protein samples obtained directly from tissue or cell media or cell media is extremely difficult work. Many biologically significant proteins are present in minute quantities in living organisms, and can be produced using in vitro methods only in limited and minute quantities.
One conventional technique for protein identification is mass spectrometry (MS) as a detection and quantification tool. Mass spectrometry is often used because it is more or less universally amenable to diverse analytes, and provides large amounts of useful analytical information from relatively small amounts of biological sample.
Mass spectrometry is conventionally used in biological research and development activities to identify unknown compounds, or to examine the structure or abundance of certain compounds. Simply put, a mass spectrometer is a detector that is capable of identifying the molecular mass of constituents within a mixture. The mass spectrometer produces a mass spectrum that can be used to identify unknowns or to determine the basic building blocks that constitute a molecule's structure.
Conventional MS identification techniques typically rely on four steps:                1) The analyte (e.g. protein or peptide) is separated from its complex biological matrix and converted to an analyzable species. Typical methods used in this step may involve any combination of such techniques as gel electrophoresis, adsorption chromatography, size exclusion chromatography, immunoprecipitation, blotting, osmosis, and chemical labeling, amongst many others. The methodology chosen for a given application typically depends on the nature of the analyte, its matrix, and the type of information required from the investigation.        2) The sample is ionized to produce gas-phase molecular ions. The ionization method may be electrospray, nanospray, atmospheric pressure chemical ionization (APCI), or matrix assisted laser desorption/ionization (MALDI), amongst others. In conventional methods, electrospray, nanospray, and MALDI are utilized for the analysis of proteins and peptides.        3) The analyte's molecular ion is introduced into a high vacuum and fragmented to produce ions. The number and size of the “daughter” ions produced are determined by the structure of the analyte and the fragmentation method. Typically, molecular ions are fragmented inside the mass spectrometer by colliding with them with a low-pressure gas.        4) The molecular fragments are sorted according to molecular mass and detected, producing a fingerprint of the original protein. This fingerprint can be used to determine characteristics (e.g., identity, quantity, or structure) of the unknown protein.        
The methods used in step 1 are also conventional in most other fields of biological and chemical analysis and are not limited in scope to MS detection methods.
For complex and more difficult protein separation and analysis, the ionization procedure in step 2 often employs nanospray techniques. The reasons for this method choice may include: 1) very small sample volumes typically require low flow rate analytical methods for efficient analysis, 2) the nanospray procedure is typically amenable to established chromatographic methods (albeit at lower flow rates), and 3) the efficiency of sample ionization increases with reduction in flow rate (thus increasing the sensitivity of analysis for low-abundance proteins/peptides).
In conventional nanospray applications, an analyte flow stream is ionized and introduced into a mass spectrometer by passing a solution of the analyte through a very small (e.g., about 20 μm inner diameter) high voltage electrified needle. The fluid passing through the needle accepts electrical charge imparted by the applied voltage, and emerges from the open end of the capillary as a finely dispersed aerosol. This charged aerosol travels at atmospheric pressure toward a counter electrode at the mass spectrometer entrance. Once the aerosol enters the mass spectrometer, it largely consists of gas-phase ions that may be analyzed using steps 3 and 4 above. Such nanospray methods require flow rates in the range of 10-1,000 nl/min, which is much lower than flow provided by conventional chromatographic methods.
In addition to mass spectrometric elucidation methods, diagnostic research has been increasingly concerned with investigating biological systems using microelectromechanical systems (MEMS). When applied to biological investigation, these methods are sometimes referred to as Lab-on-a-chip (LOC) analyses. Systemically, many LOC instruments can be viewed as incorporating nano flow, microarray, and biosensor technologies into an integrated device. In a typical approach, macroscopic analytical devices (e.g., valves, columns, detection chambers, and flow channels) are miniaturized and micro-fabricated onto glass, silicon, or polymeric material.
To date, such systems and methods utilize non-feedback based pumping solutions, including syringe and positive displacement pumps. Because of the relatively small flow rates required in such applications, many conventional systems utilize electroosmotic flow (EOF) pumping in lieu of mechanical pumps. Although the EOF pumping method is convenient to use and can operate at low average flow rates, it presents significant disadvantages when required for precise control, or when used on fluids that have high ionic strength.
In addition to the foregoing applications, worldwide experience over the past several years have heightened interest in the detection and elucidation of chemical or biological threat agents introduced into the environment through terrorist or military action.
In a conventional implementation, a point detector for such threat agents operates as a self-contained analytical module that is capable of analyzing environmental samples and initiating an alarm if hazards are found to be present above a prescribed threshold concentration. Current developmental point detection technologies rely on such conventional techniques as enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), surface plasmon resonance (SPR), cell cytometry, cell staining, immunoprecipitation, mass spectrometry, and native fluorescence, to name a few.
In order to adapt higher flow methods to nanospray, lab-on-a-chip, or other ultra-low flow methodology, two different approaches have been utilized. In the “split flow” method, a fraction of the analytical fluid is split from a conventional high-flow stream into a low-flow stream and the reduced flow stream is applied to the analytical method. Equipment designed to do this are known as splitflow instruments. In the typical conventional split flow configuration, the flow stream is passed into a tee connector with different backpressures generated on each outlet arm of the tee. One arm becomes the high-flow arm and the other becomes the low-flow arm. The ratio of high to low flow is determined by the pressure ratio between the two arms. Flow split ratio can be adjusted by changing tubing lengths or diameters (thus adjusting the pressure generated at a given flow rate) on each arm.
The two largest disadvantages of the split flow technique are that most of the analytical solvents is discarded in the process, and that high precision is very difficult (or impossible) to obtain at low flow rates. Imprecision in flow rates can easily result from changes in temperature, particulates in the fluid, or gradual changes in backpressure created from downstream devices.
In “splitless” methods, the chromatographic and upstream fluid systems are converted to low flow technologies. This method requires pumping and fluid transfer systems that are capable of operating at rates from 10-1,000 nl/min. Such methods are deemed splitless flow. The main disadvantages of this method are the requirements for precise control of ultra-low flow rates and the requirement for very low dead volume flow paths in the instrumentation. The splitless configuration has the advantage of very low solvent consumption, and is directly amenable to injection and handling of very small samples.
Analytical methods and systems have been developed that demand sensitive high-throughput analyses of biological materials in small quantities. Often, such analyses require precise control of the fluid flow rates in the range of about one (1) nano-liter (nL) per minute to about five (5) microliters (μL) per minute, with pressures varying over a range of several orders of magnitude. Such analytical applications include, among others, nano-scale liquid chromatography (nano-LC), mass spectrometry (MS), or capillary electrophoresis (CE). These microfluidic applications typically utilize fluid flow rates as low as tens of nanoliters per minute up to several microlitres per minute. Designing systems to precisely achieve and maintain ultra-low flow rates is a difficult task, fraught with several potential problems.
One problem affecting such microfluidic techniques comes from the susceptibility of various components of systems used for conventional ultra-low flow applications to compress or decompress in response to a change in system pressure. This component adjustment to pressure change often creates a significant delay time before achieving a desired flow rate in conventional microfluidic systems and applications, and can also hinder accurate flow rate adjustment in such systems and applications.
Another persistent problem with such conventional microfluidic systems and applications occurs when air or other gases are inadvertently entrained into the flow path of such a system. If these compressible gases are present in the flow path of conventional systems for such applications, the compression and expansion of gas bubbles creates difficulties in achieving a desired flow rate.
In many conventional microfluidic systems, the flow rate of a fluid is established in a pump by displacing liquid at a controlled rate using, for example, a piston or syringe plunger. To obtain desired flow rates in such conventional systems, the displacing element of the pump is moved at a fixed velocity using a preprogrammed control system. Such conventional systems often show undesirable flow rate fluctuations created from imprecision in the mechanical construction of the drive system used to displace the liquid. In conventional lead screw-driven systems, for example, inaccuracies often arise from periodic changes in screw characteristics as the screw turns through a complete revolution, and from inaccuracies in thread pitch along the screw, among other types of mechanical errors.
In order to overcome these difficulties in achieving and maintaining desired flow rates, conventional flow sensors may be employed to allow the system to compensate for inaccuracies through use of a feedback loop to a preprogrammed control system. Many conventional flow sensors used in microfluidic analysis, such as the SLG1430 sensor that is commercially available from Sensirion Inc. (of Zurich, Switzerland), have a non-linear response to fluid flow. For such flow sensors, the sensor response to increasing flow rate approximates a polynomial equation, with the equation order and constants dependent on variables such as flow sensor design, the liquid that is being monitored, and the operating flow rate range.
In order to use such conventional flow sensors to measure and maintain accurate ultra-low flow rates in conventional systems via a feedback loop, the sensor must be calibrated for the solvent that is to be passed through the sensor. Conventional calibration methods usually involve preparation of a list of the sensor responses at different flow rates for a given solvent. When a particular solvent is used, the actual flow rate is obtained by comparing the sensor response to tabulated calibration values gathered from repeated observations made for that particular sensor and solvent combination. Calibration curves for a given sensor and solvent can be obtained by fitting the calibration data to a best-fit curve from the empirical data in such conventional calibration methods.
A major problem with this conventional calibration tabular methodology is that data values must be collected for any solution mixture that is to be passed through the system. Doing so for numerous solvents can require a significant amount of time and effort. Moreover, for reliable operation, this data must be collected using a precise flow rate reference. Often, a conventional microfluidic system will be used to deliver different solutions that possess diverse characteristics, and calibrating a conventional system for these various solutions is often time consuming and laborious.
In conventional chromatograph applications, analytical columns consist of narrow-bore tubes made of fused silica, polymeric material, stainless steel, or other material that should be compatible with the analyte mixture and mobile phase. The columns may be one centimeter to several meters long, and they are typically packed with small beads ranging in size from a few microns to several millimeters in diameter. The tubes may have fritted on the downstream end to prevent loss of beads when liquid is flowed through. Long-chain carbon polymers are coated on the beads to comprise the stationary phase in conventional reverse-phase separations. A sample is introduced into the head of the column using an injection valve. Detection is performed using UV absorbance, fluorescence, mass spectrometry, emission spectroscopy, nuclear magnetic resonance (NMR), or some other method that is sensitive to the analyte in question.
When the analyte compounds are similar in nature, then the mobile phase mixture can remain constant throughout the entire separation. This type of separation is known as isocratic. For more complex mixtures that contain diverse compounds covering a range of hydrophobicity, increasing the organic content of the mobile phase during the separation may be desired. This later method is known as gradient chromatography.
In a conventional reverse-phase gradient chromatography analysis, the amount of organic modifier in the mobile phase may be increased from the beginning of the analysis until elution of the most hydrophobic compounds. This increase in hydrophobic nature of the mobile phase serves to enhance elution of very highly retained compounds, but allows the weakly-retained compounds to separate under low organic modifier concentrations.